Processes of thickening and of oil recovery using polysaccharide polymer made by xanthomonas

ABSTRACT

A polysaccharide polymer is disclosed which is a better viscosifier of water than xanthan gum. The polysaccharide polymer and its non-acetylated form, are comprised of glucose and mannose moieties in a ratio of about 2:1. The invention also discloses Xanthomonas mutants which produce the polysaccharide polymer but which do not produce xanthan gum. Methods of preparing the polysaccharide polymers and of their use are also described.

This application is a continuation of application Ser. No. 07/333,285,filed Apr. 5, 1989, now abandoned, which is a divisional of Ser. No.07/099,618, filed Sept. 22, 1987, now U.S. Pat. No. 4,868,293 which is adivisional of Ser. No. 06/762,878, now U.S. Pat. No. 4,713,449, filedAug. 6, 1985.

BACKGROUND OF THE INVENTION

Xanthan gum is produced by bacteria of the genus Xanthomonas, such asthe species campestris, albilineans, fragaria, vesicatoria, and thelike. Xanthan gum is a widely used product due to its unusual physicalproperties: extremely high specific viscosity and pseudo-plasticity. Itis commonly used in foods as a thickening agent and in secondary oilrecovery as mobility control and profile modification agents and inpetroleum drilling fluids.

Chemically, xanthan gum is an anionic heteropolysaccharide. Therepeating unit of the polymer is a pentamer composed of five sugarmoieties: two glucose, one glucuronic acid and two mannose moieties.They are arranged such that the glucose moieties form the backbone ofthe polymer chain, and side chains of mannose-glucuronic acid-mannosegenerally extend from alternate glucose moieties. Often this basicstructure is specifically acetylated and/or pyruvylated. (Janson, P. E.,Kenne, L., and Lindberg, B., Carbohydrate Research, 45, 275-282 (1975);Melton, L. D., Mindt, L., Rees, D. A., and Sanderson, G. R.,Carboyhydrate Research, 46, 245-257 (1976).) This and all otherpublications referred to herein are specifically incorporated byreference. The structure is depicted below: ##STR1##

In spite of the broad utility of naturally occurring xanthan gum, thereare some situations where its physical properties become limiting. Inparticular, in secondary oil recovery it is not uncommon for thetemperature of the oil-bearing reservoir and salt concentrations in thereservoir brine to be higher than are optimal for xanthan solutions.When these conditions occur, xanthan can precipitate, flocculate and/orlose viscosity. Therefore there is a need for new viscosifying productswhich perform well at high temperature and high salt conditions.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a polysaccharidepolymer which is a better viscosifier of water than naturally occurringxanthan gum.

It is a further object of this invention to provide a polysaccharidepolymer having improved rheological properties over naturally occurringxanthan gum at elevated temperature and/or in the presence of salts.

It is a further object of the present invention to provide amicroorganism having the ability to produce the polysaccharide polymer.

It is a further object of the invention to provide a process forpreparing the polysaccharide polymer by aerobically fermenting amicroorganism having the ability to produce the polysaccharide polymer.

In accordance with this invention, there is provided a compositioncomprising a polysaccharide polymer containing essentially no glucuronicacid moieties having a D-glucose: D-mannose ratio of about 2:1, whereinthe D-glucose moieties are linked in a beta-[1,4] configuartion to formthe polymer backbone, and the D-mannose moieties are each linked in analpha-[1,3] configuration generally to alternate glucose moieties. Theinvention also contemplates processes for making the polysaccharidepolymer, microorganisms which make the polysaccharide polymer, andmethods of using the polysaccharide polymer.

The polysaccharide polymer of this invention can be made by blocking oneof the steps in xanthan gum biosynthesis. Therefore, rather than havinga three-sugar side-chain extending from the backbone ofbeta-[1,4]-D-glucose as in xanthan gum, the polysaccharide polymer ofthis invention has a single sugar moiety generally linked to alternateglucose moieties of the backbone. The polysaccharide polymer of thisinvention is herein termed "polytrimer" because it consists of arepeating trimer unit, glucose-glucose-mannose. Its structure is shownbelow, where n is the number of repeating units in the polymer. ##STR2##As shown by the above, the polytrimer consists of D-mannose linkedalpha-[1,3] generally to alternate moieties of beta-[1,4] linkedD-glucose. As in xanthan gum, an acetic acid moiety can be, but is notalways, esterified at the 6-O position of mannose, as described inSutherland, I. W., Carbohydrate Polymers, 1, 107-115, (1981). Althoughthe structure of the polysaccharide polymer is thought to be as shown,it is possible that under certain conditions of synthesis, a mannosemoiety may not always be linked at alternating glucose residues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the assumed pathway of xanthan gum biosynthesis. It isbased on the data of several laboratories. See, Ielpi, L., Couso, R. O.,and Dankert, M. A., Biochem. Biophy. Res. Comm., 102, 1400-1408 (1981),FEBS Letters, 130, 253-256 (1981), Biochem. Intern., 6, 323-333 (1983);Osborn, M. J. and Weiner, I. M., J. Biol. Chem., 243, 2631-2639 (1967);Troy, F. A., Annual Reviews of Microbiology, 33, 519-560 (1979).Abbreviations used are: glu=glucose, gluA=glucuronic acid, man=mannose,glu-glu=cellobiose, P=phosphate, PP=pyrophosphate, C55=isoprenoid lipidcarrier, PEP=phosphoenolpyruvate, AcCoA=acetyl coenzyme A,I-V=glycosyltransferases, UDP=uridine 5'-diphosphate, GDP=guanosine5'-diphosphate.

FIG. 2 shows the viscosities of solutions of polytrimer and xanthan gum,each at 1000 ppm in 10 weight percent NaCl brine, as a function of shearrate.

FIG. 3 shows the ratio of viscosities of solutions of 1,000 ppmpolytrimer to xanthan gum as a function of brine salinity.

FIG. 4 shows the ratio of viscosities of solutions of polytrimer toxanthan gum as a function of polymer concentration in 10 weight percentNaCl brine.

FIG. 5 shows the ratio of viscosities of solutions of 1,000 ppmpolytrimer to xanthan gum as a function of temperature in brines ofvarious salinities.

DETAILED DESCRIPTION OF THE INVENTION

The polysaccharide polymer of this invention can be made with acell-free enzyme system or can be made by growing cells of anappropriate mutant strain. Other means of preparing the polysaccharidepolymer are also described below.

The basic method relating to the use of a cell-free system to makexanthan gum is described in Ielpi, L., Couso, R. O., and Dankert, M. A.(FEBS Letters, 130, 253-256, (1981)) and can also be employed to makethe polysaccharide polymer of this invention. For example, wild-typeXanthomonas campestris cells can be lysed by a freeze-thaw process andthe substrates for polytrimer synthesis, UDP-glucose and GDP-mannose,with or without acetyl-CoA, can be added to the lysate. Alternate meansof lysis may be used including but not limited to sonication, detergenttreatment, enzyme treatment and combinations thereof. The lysate may beused in its crude form, or purification of the enzymes may be employed.The enzymes of the xanthan gum biosynthetic pathway covalently join theglucose and mannose moieties as in the normal pathway. Since the enzymeshave no UDP-glucuronic acid to add to the nascent chains, the pathway isblocked at reaction IV (see pathway, FIG. 1,) and the intermediateisoprenoid lipid- pyrophosphate-glucose-glucose-mannose accumulates.Surprisingly, the xanthan polymerase which ordinarily acts onlipid-linked pentamer (glucose-glucose-mannose-glucuronic acid-mannose)is able to polymerize lipid-linked trimer, (glucose-glucose-mannose.)Thus, the polytrimer of the present invention can be synthesized invitro.

The cell-free synthesis of polytrimer described above shows thatXanthomonas campestris cells have all the enzymes necessary tosynthesize polytrimer. However, to use whole cells to synthesizepolytrimer in vivo, a means of blocking xanthan gum synthesis atreaction IV (see FIG. 1) is required. Mutagenesis can be employed toblock reaction IV.

Transposons, including but not limited to Tn10 and Tn903, can be used tomutagenize Xanthomonas. These transposons confer resistance totetracycline and kanamycin, respectively. Transposons have the abilityto insert themselves into genes; when they do so, they cause mutationsby interrupting the coding sequence, (Kleckner, N., Annual Reviews ofGenetics, 15, 341 (1981).) The transposons can be introduced intoXanthomonas on a so-called suicide vector, such as pRK2013. This vectorhas the ability to transfer itself into non-enteric bacteria, such asXanthomonas, but cannot maintain itself (replicate) in that host,(Ditta, G., Corbin, D., Helinski, D. R., Proc. Natl. Acad. Sci. USA, 77,7347-7351 (1980). Thus, if the suicide vector is introduced into apopulation of Xanthomonas cells, and that population is subsequentlychallenged with either tetracycline or kanamycin, the individuals whichsurvive are those in which one of the transposons has inserted itselfinto the genome of Xanthomonas. Survivors of such a challenge can bescreened for those which have lost the ability to make xanthan gum. Suchmutants appear less mucoid than wild-type Xanthomonas.

In other embodiments of the invention, other means of mutagenesis can beemployed to generate mutants which have lost the ability to make xanthangum. Such means will readily occur to one skilled in the art, andinclude, without limitation, irradiation, recombinant DNA technology,and chemical mutagen treatment (Miller, J. H., Experiments in MolecularGenetics (1972); Davis, R. W., Botstein, D., and Roth, J. R., AdvancedBacterial Genetics (1980); Maniatis, T., Fritsch, E. F., Sambrook, J.,Molecular Cloning (1982), Cold Spring Harbor).

Although mutants can first be chosen which appear less mucoid thanwild-type, those desired retain the ability to make some polysaccharide.Cell-free extracts of each of the xanthan gum deficient mutants can beprepared and tested by adding different combinations of substrates andanalyzing the products. For example, if UDP-glucose, GDP-mannose, andUDP-glucuronic acid are added as substrates, the product should beidentical to that produced when UDP-glucose and GDP-mannose are added.Alternatively, appropriate mutants can be detected by assaying theculture broth of each mutant for the presence of polytrimer. Thusxanthan gum deficient mutants can be found which appear to be blocked atreaction IV of the xanthan gum pathway. A mutant of this description hasbeen placed on file at the American Type Culture Collection, Rockville,Md., as ATCC No. 53195. Such mutants can be used to synthesizepolytrimer in vivo.

Although glycosyltransferase IV mutants have been employed in theexamples to make the polytrimer of the present invention, otherembodiments of the invention contemplate use of mutants inUDP-glucuronic acid metabolism. Such a mutant has been isolated anddeposited at the American Type Culture Collection, Rockville, Md., underthe ATCC No. 53196.

It is not beyond the scope of the invention to employ an enzymeinhibitor of wild-type glycosyltransferase IV or of UDP-glucuronic acidbiosynthesis to arrive at the same product. Still other alternatives forproducing polytrimer are contemplated including enzymatic and chemicaldegradation of natural xanthan gum as, for example, by removing theterminal mannose and glucuronic acid moieties from the side chains ofxanthan gum.

Using similar schemes to mutagenize strains of Xanthomonas, it ispossible to obtain mutants which produce other new polysaccharidepolymers. For example, a mutation in the acetylase gene yieldscompletely non-acetylated xanthan gum. When an acetylase mutation and aglycosyltransferase IV mutation are put in the same strain (a doublemutant), a non-acetylaled polytrimer is produced. Other mutations andcombinations of mutations of the xanthan pathway are possible to yieldnew products.

The mutants can be grown under conditions known in the art for growth ofwild-type Xanthomonas. For example, they can be grown on suitableassimilable carbon sources such as glucose, sucrose, maltose, starch,invert sugar, complex carbohydrates such as molasses or corn syrup,various organic acids and the like. Mixtures of carbon sources can alsobe employed. The concentration of carbon source supplied is oftenbetween about 10 and 60 grams per liter. Also necessary for growth arean assimilable source of organic or inorganic nitrogen, generallybetween about 0.1 and 1.0 grams per liter, and minerals, the choice ofwhich are easily within the skill of the art. Examples of suitablenitrogen sources are ammonium salts, nitrate, urea, yeast extract,peptone, or other hydrolyzed proteinaceous materials or mixturesthereof. Examples of suitable minerals include phosphorous, sulfur,potassium, sodium, iron, magnesium; these are often added with achelating agent such as EDTA or citric acid.

Optimal temperatures for growth of Xanthomonas generally are betweenabout 18° and 35° C., preferably between about 28° and 32° C.Xanthomonas cells are grown aerobically by supplying air or oxygen sothat an adequate level of dissolved oxygen is maintained, for example,above about 10% of saturation. Preferably the level is kept above about20%. The pH often is maintained at about 6.0 to 8.0, preferably at about6.5 to 7.5.

The polysaccharide polymer of the present invention can be recoveredfrom fermentation broths by a suitable means. Precipitation withisopropanol, ethanol or other suitable alcohol readily yields thepolytrimer gum. Generally, alcohols are added to a concentration ofabout 50 to 75%, on the basis of volume, preferably in the presence ofpotassium chloride, sodium chloride or other salt. Alternatively, thepolymer can be recovered from the broth by ultrafiltration.

When chemical analyses are performed on polytrimer gum to determine theratio of glucose:mannose, a variation from the theoretical value of 2:1is found. The same type of variation is found when analyzing xanthangum. Measured ranges of the ratio of glucose: mannose will generally bebetween about 1.4:1 and about 2.4:1. Preferably the ratio will bebetween 1.7:1 and 2.1:1.

Levels of acetylation of the mannose residues of the polysaccharidepolymer vary. In addition, it is not beyond the scope of the inventionto employ a microorganism to make the polysaccharide polymer which isincapable of acetylating the mannose residue, such asacetylase-deficient mutants. In such a case there will be no acetylatedmannose residues in the polysaccharide polymer.

Typically, concentrations of polytrimer in the fermentation broth areabout 0.1% (w/w). Routine testing of fermentation conditions andclassical and recombinant DNA strain improvement techniques, all withinthe skill of the art, can be employed to improve the yield.

On a weight basis, polytrimer is superior to xanthan as a viscosifier ofan aqueous medium. The viscosity of solutions of polytrimer is retainedat conditions of high temperatures and/or high salinity. Such solutionscan be prepared at any desirable concentrations, preferably betweenabout 0.01% and about 15%, by dissolving the polysaccharide polymer inan aqueous medium. The product of this invention is ideally suited foruse in secondary oil recovery. The same techniques as are used withxanthan gum in the art, and are well-known is secondary oil recovery,are appropriate with the polysaccharide polymer. See, for example,Lindblom, G. P., et al., U.S. Pat. No. 3,198,268.

Mobility control solutions for use in enhanced oil recovery can beprepared from the polysaccharide polymer. Concentrations of from about100 to about 3,000 ppm of the polysaccharide polymer are appropriate forsuch mobility control solutions. Other known additives may also be usedin, or in combination with, these solutions to further enhance oilrecovery. Such additives include, for example, surfactants and alkalineagents.

The polysaccharide polymer, like xanthan gum, can also be used as athickening agent in foods, cosmetics, medicinal formulations, papersizings, drilling muds, printing inks, and the like. In addition it canbe used to reduce frictional drag of fluid flow in pipes.

The following examples are provided by way of exemplification and arenot intended to limit the scope of the invention.

EXAMPLE 1

This example shows how the product of the present invention can beprepared in vitro, and identifies it as a truncated product of thexanthan pathway.

Preparation of Lysates

Xanthomonas campestris B1459 S4-L was obtained from Northern RegionalResearch Laboratories of the U.S. Department of Agriculture. Bacteriawere grown in YM (yeast-malt medium) supplemented with 2% (w/v) glucoseas described by Jeanes, A., et al. (U.S. Department of Agriculture,ARS-NC-51, 14 pp (1976)). Cultures were grown to late log phase at 30°C. at 300 rpm. The cells were titered on YM plus 2% (w/v) glucose platesat 30° C. The cells were harvested by centrifugation and washed withcold Tris-HCl, 70 mM, pH 8.2. Washed cells were resuspended in Tris-HCl,70 mM, pH 8.2 with 10 mM EDTA and were freeze-thawed three times by aprocedure similar to Garcia, R. C., et al. (European Journal ofBiochemistry 43, 93-105, (1974)). This procedure ruptured the cells, aswas evidenced by the increased viscosity of the suspensions and thecomplete loss of cell viability (one in 10⁶ survivors) after thistreatment. The freeze-thawed lysates were frozen in aliquots at -80° C.Protein concentration was determined with BIO RAD assay (BIO RADLaboratories, Richmond, Calif.) and was found to be 5 to 7 mg cellprotein per ml of lysate.

Biosynthetic Assay Procedure

As described in Ielpi, L., Couso, R. O., and Dankert, M. A., FEBSLetters, 130, 253-256 (1981), an aliquot of freeze-thawed lysate(equivalent to 300 to 400 ug protein), DNAase I (10 ug/ml), and MgCl₂ (8mM) were preincubated at 20° C. for twenty minutes. An equal volume of70 mM Tris-HCl, pH 8.2, with the desired radiolabeled sugar nucleotides(UDP-glucose and GDP-mannose), with or without UDP-glucuronic acid, wasadded and incubated at 20° C. At various times, the reactions werestopped by the addition of EDTA to 4 mM. The samples were centrifuged;the pellets were washed two times with buffer. The supernatants werecombined, carrier xanthan (100 ug) was added, and the xanthan plussynthesized polymer were precipitated with ethanol(60%)-KCl(0.8%). Theprecipitated polymer was resuspended in water and reprecipitated twomore times to remove unicorporated label. Radioactivity incorporatedinto the gum fraction was determined in a liquid scintillation counter,and the data were processed to obtain incorporation in terms of pmoles.

                  TABLE 1                                                         ______________________________________                                        Incorporation of labeled sugars by freeze-thaw cell lysate                    of X. campestris B1459 S4-L into gum                                                        Gum Fraction (pmol)                                             Incubation Mix  [.sup.3 H]man                                                                           [.sup.14 C]glc                                                                         glc/man                                    ______________________________________                                        +UDPG, GDPM      98        201     2.1                                        +UDPG, GDPM, UDP-GA                                                                           1540      1562     1.0                                        dpm/pmol .sup.3 H = 442                                                       .sup.14 C = 37.5                                                              ______________________________________                                         UDPG = UDPglucose                                                             GDPM = GDPmannose                                                             UDPGA = UDPglucuronic acid                                                    dpm = disintegrations per minute                                              pmol = picomole                                                               glc = glucose                                                                 man = mannose                                                            

Cell lysates of B1459 S4-L were incubated at 20° C. for 30 minutes andprocessed to give the gum fractions as described in the text. The molarratio of glucose to mannose is the ratio of pmoles of incorporatedcarbon-14 to tritium labeled sugars in the gum fractions.

In the presence of all three sugar constituents, the ratio of glucose:mannose was 1.0:1, as expected for xanthan gum. When UDP-glucuronic acidwas absent, the ratio was 2.1:1. See Table 1. This ratio is consistentwith the hypothesis that the polysaccharide polymer is formed of trimerunits which are intermediates in the xanthan gum biosynthetic pathway.

A pulse-chase in vitro experiment showed that lipid-linked cellobiose (aglucose dimer) was processed to lipid-linked trimer(glucose-glucose-mannose) and subsequently to polytrimer gum. Afreeze-thaw lysate of strain B1459 S4-L was prepared as described above.UDP-[¹⁴ C]glucose was added to the lysate, comprising the "pulse", andradiolabeled cellobiose accumulated on the lipid carrier during anincubation of 13 minutes. The "chase" consisted of addition of 100-foldexcess unlabeled UDP-glucose as well as GDP-[³ H]mammose. Aliquots ofthe incubation mixture of lysate and sugar nucleotides were removed atvarious times and processed to produce an organic extract (lipidcarrier-linked fraction) and an aqueous fraction (containing gum). Theoligosaccharides of the organic extract were acid hydrolyzed from thelipid carrier, dephosphorylated, separated by thin layer chromatography,removed from the chromatograms and the radiolabel quantitated. Theresults are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Fate of UDP-[.sup.14 C] glucose in pulse-chase in vitro experiment            with cell lysates of B1459 S4-L                                               ______________________________________                                        Pulse (12 min)                                                                              9     pmol    Lipid-linked cellobiose                           Chase (4 min) 1     pmol    Lipid-linked cellobiose                                         10    pmol    Lipid-linked trimer                               Chase (16 min)                                                                              0.5   pmol    Lipid-linked cellobiose                                         6     pmol    Lipid-linked trimer                                             3     pmol    Soluble polytrimer                                Chase (48 min)                                                                              0.2   pmol    Lipid-linked cellobiose                                         0.4   pmol    Lipid-linked trimer                                             10    pmol    Soluble polytrimer                                ______________________________________                                    

The experimental conditions and the processing of the organic fractionand the soluble gum fraction are described in the text of Example 1.

The labeled glucose from UDP-[¹⁴ C]glucose, as can be seen in Table 2,was immediately incorporated into lipid-linked cellobiose in the"pulse". Upon addition of GDP-mannose and excess UDP-glucose (thechase), the labeled cellobiose was converted rapidly to labeledlipid-linked trimer, which was later detected as polytrimer gum in theaqueous fraction, at about 16 minutes after the chase began. Thisdemonstrates the precursor-product relationships of UDP-glucose,lipid-linked cellobiose, lipid-linked trimer, and polytrimer gum, andtheir relationships to the xanthan biosynthetic pathway.

EXAMPLE 2

This example demonstrates the molar ratio of glucose to mannose inpolytrimer gum synthesized in vitro by a glycosyltransferaseIV-deficient mutant.

The method of preparing the lysate is described above in Example 1. Thestrain used to prepare the lysate was that designated ATCC No. 53195.Added to the lysate were either 1, 2 or 3 nucleotide-charged sugars,consisting of UDP-[¹⁴ C]glucose alone, UDP-[¹⁴ C]glucose and GDP-[³H]mannose, or UDP-[¹⁴ C]glucose, GDP-[³ H]mannose and unlabeledUDP-glucuronic acid. At 30 minutes after addition of the sugarsubstrates, the aqueous fraction was processed and analyzed as describedin Example 1. Results are shown in Table 3. When two sugar substrates,UDP-glucose and GDP-mannose, were present in the incubation mixture themolar ratio of glucose to mannose found in the gum was 2.4:1. When allthree sugar substrates were incubated together with the lysate, theresulting gum had a 2.3:1 molar ratio of glucose to mannose.

                  TABLE 3                                                         ______________________________________                                        Incorporation of labeled sugars by freeze-thaw cell lysate                    of ATCC No. 53195 into polytrimer gum                                                        Gum Fraction (pmol)                                            Reaction Mix     [.sup.3 H]man                                                                          [.sup.14 C]glc                                                                         glc/man                                    ______________________________________                                        +2 UDPG, GDPM    71       174      2.4                                        +3 UDPG, GDPM, UDP-GA                                                                          65       152      2.3                                        dpm/pmol .sup.3 H = 340                                                       .sup.14 C = 40                                                                ______________________________________                                         Abbreviations are explained in legend to Table 1.                        

Cell lysates of ATCC No. 53195 were incubated at 20° C. for 30 minutesin the reaction mixes indicated and processed to give the gum fractionsas described in Example 1. The molar ratio of glucose to mannoseindicated is the ratio of pmoles of incorporated carbon-14 to tritiumlabeled sugars in the processed fractions.

The presence of UDP-glucuronic acid has no effect on the ratio ofglucose to mannose incorporated into a polysaccharide polymer when thecell-free lysate used is from a glycosyltransferase IV-deficient mutant.The biochemical phenotype of the mutant lysate when incubated with allthree sugars is analogous to that of the wild-type lysate when incubatedwith only two sugar substrates, in that the in vitro produced gums bothhave a molar ratio of approximately 2:1 of glucose to mannose moieties.

EXAMPLE 3

This example demonstrates that the trimeric intermediate which ispolymerized to form polytrimer gum has the same anomeric configurationof the sugars as in xanthan gum. In addition it demonstrates that themannose of the trimer is attached to the non-reducing glucose ofcellobiose in the lipid-linked intermediate.

Alpha-mannosidase (EC 3.2.1.24) and beta-glucosidase (EC 3.2.1.21) wereused to singly or sequentially treat the trimeric oligosaccharide whichhad been synthesized and double labeled in vitro as described inExample 1. Alpha-mannosidase will hydrolyze terminal, unsubstitutedmannose residues attached through an alpha-1 linkage. Beta-glucosidasewill hydrolyze terminal, unsubstituted D-glucosyl residues attached in abeta-1 linkage.

The trimer was removed from the lipid and dephosphorylated. This wasthen deacetylated by base treatment, such as pH12 for 2 to 3 hours,because alpha-mannosidase cannot recognize acetylated mannose moieties.

The results were as follows. Treatment of trimeric oligosaccharide withbeta-glucosidase left it unchanged. When alpha-mannosidase was used totreat the trimeric oligosaccharide, cellobiose and mannose were formed.When the trimeric oligomer was treated with alpha-mannosidase, first,and beta-glucosidase, second, glucose and mannose were formed. Theresults confirm that mannose is attached to the non-reducing glucose byan alpha-linkage in the trimeric intermediate, and that the glucosemoieties are beta-linked. This confirms that trimer is an intermediateproduct of the normal xanthan enzyme pathway.

EXAMPLE 4

This example shows the methods of mutagenesis and screening which wereemployed to generate the mutant strains which are xanthan gum deficientdue to a lesion in the gene for glycosyltransferase IV.

Xanthomonas campestris, genetically marked with a chromosomal resistanceto streptomycin, was used as a recipient in a conjugation with E. coliLE392 containing plasmid pRK2013::Tn10. Plasmid pRK2013 contains Tn903which encodes kanamycin resistance, (Figurski, D. H., and Helinski, D.R., Proc. Natl. Acad. Sci., U.S.A., 76, 1648-1652 (1979),) and theplasmid cannot replicate in Xanthomonas, (Ditta, G., et al., supra.)Transposon Tn10 encodes resistance to tetracycline. Transconjugants wereselected which were resistant to streptomycin and kanamycin, orstrptomycin and tetracycline. The former occurred at a frequency ofabout 4×10⁻⁶ /recipient and presumably resulted from a transposition ofTn903. The latter occurred at a frequency of about 3×10⁻⁶ /recipient andpresumably resulted from a transposition of Tn10 into the genome ofXanthomonas campestris.

Auxotrophs were found among these transconjugants at a frequency ofabout 2%; their needs were widely distributed among the variousnutritional requirements. This indicates that these transposons do nothave a particularly preferred locus for insertion in Xanthomonas.Prototrophic revertants of the auxotrophs were selected, and most werefound to be drug-sensitive; this suggests that the auxotrophies werecaused by transposon insertion.

To screen for xanthan gum deficient mutants among the doubly resistanttransconjugants, Congo Red dye, which enhances the morphologicaldistinction between xanthan gum producing and non-producing colonies,was added to the solid media. Colonial morphology was examined after 7to 12 days incubation at 30° C. Xanthan gum deficient mutants were foundat a frequency of approximately 10⁻⁴.

To identify a glycosyltransferase IV mutant from among the xanthan gumdeficient mutants, freeze-thaw lysates of each were prepared.Radiolabeled UDP-glucose and GDP-mannose were added with or withoutUDP-glucuronic acid. The desired mutants made a gum having aglucose:mannose ratio of about 2:1, irrespective of the presence ofUDP-glucuronic acid. Several mutants were found of this description.They contain lesions due to Tn10 insertion. Mutants induced by Tn903were also found having this phenotype. In addition mutants have beenisolated having this phenotype which were induced by nitrosoguanidine.

EXAMPLE 5

This example demonstrates the use of a glycosyltransferase IV deficientmutant to produce polytrimer gum in vivo.

To obtain in vivo synthesized gums, five liters each of wild-type NRRLB-1459 S4-L and the glycosyltransferase IV deficient mutant of Example 4(ATCC No. 53195) were aerobically grown in a fermenter at 28° C. to 32°C., with the pH controlled at pH 6.0 to 8.0. A minimal medium was usedcontaining 10 g/l potassium phosphate, 1.43 g/l ammonium sulfate, 2 g/lcitric acid, 30 g/l glucose, and trace elements. After 145 hours, thegums were recovered and purified. The cells were removed bycentrifugation and the gums precipitated from the broth by addition ofisopropanol (55% v/v) and sodium chloride (0.5% w/v). The precipitateswere collected by filtration and redissolved in water. The gums werereprecipitated with isopropanol (55% v/v) without salt and redissolvedin water. The preparations were dialyzed using 12,000 MW cutoff membranedialysis tubing against water for three days.

The glucose:mannose ratios were determined by complete acid hydrolysisof the polysaccharide polymers with subsequent analysis by highperformance liquid chromatography (HPLC), and found to conform to theratios found for the in vitro synthesized polymers. Theglycosyltransferase IV deficient mutant designated ATCC No. 53195 made agum with a glucose to mannose ratio of about 2.15:1, whereas thewild-type made a gum of ratio about 0.96:1.

Other in vivo produced samples of polytrimer gum were assayed by HPLC orby enzymatic analyses of the sugars after acid hydrolysis. For thetwenty-four analyses performed, the molar ratios range from 1.43:1 to2.44:1 of glucose to mannose. The mean ratio was 1.90±0.15:1 forpolytrimer made by the glycosyltransferase IV deficient mutant strain.

Also shown by the HPLC analysis of the in vivo produced polytrimerwithin detectable limits were: 1-the absence of glucuronic acid; 2-theabsence of pyruvate; 3-the presence of acetate; 4-the absence of sugarsother than glucose and mannose.

EXAMPLE 6

This example shows that polytrimer provides aqueous solutions whichexhibit improved rheological properties compared to xanthan gum over arange of temperatures and inorganic salt concentrations.

Solutions of polytrimer gum (synthesized in vivo in accordance withExample 5) and xanthan gum (purified Pfizer Flocon 4800, were preparedat a concentration of 1,000 ppm in a water containing 10 weight percentsodium chloride. Polytrimer gum shows substantially greater viscositythan xanthan gum over a wide range of shear rates (FIG. 2).

The ratio of polytrimer to xanthan viscosity at room temperature varieswith water salinity and is between 2 and 2.5 over a salinity range of 0to 20 weight percent sodium chloride, as shown in FIG. 3. The ratio ofpolytrimer viscosity to xanthan viscosity also varies with polymerconcentration (FIG. 4). Finally, the improvement in polytrimer viscosityover xanthan viscosity increases with temperature over a range of 25° to75° C., for water salinities of 0 to 20 weight percent sodium chloride(FIG. 5).

Since variations of this invention will be apparent to those skilled inthe art, it is intended that this invention be limited only by the scopeof the claims.

What is claimed is:
 1. A process for increasing the viscosity of anaqueous medium comprising dissolving a polysaccharide polymer containingessentially no glucuronic acid moieties, having a D-glucose: D-mannoseratio of about 2:1, wherein the D-glucose moieties are linked in abeta-(1,4) configuration, and the D-mannose moieties are generallylinked to alternate glucose moieties in an alpha-(1,3) configuration inan aqueous medium at a concentration sufficient to increase viscosity.2. A process for the recovery of oil form an oil-bearing subterraneanformation comprising: injecting a solution containing a polysaccharidepolymer containing essentially no glucuronic acid moieties, and having aglucose:mannose ratio of about 2:1 into a well to displace trapped oilfrom the porous rock, and collecting the displaced oil.